Oncogene (1997) 15, 1417 ± 1424 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00 Fibroblast growth factor-5 stimulates mitogenic signaling and is overexpressed in human pancreatic cancer: evidence for autocrine and paracrine actions Marko Kornmann1, Toshiyuki Ishiwata1, Hans G Beger2 and Murray Korc1 1Department of Medicine, Biological Chemistry and Pharmacology, University of California, Irvine, Medical Science Institute C240, Irvine, California 92697, USA; 2Department of General Surgery, University of Ulm, SteinhoÈvelstr. 9, 89075 Ulm, Germany Fibroblast growth factor (FGF)-1 and -2 are over- or FGF-9), and FGF-10 (Yamasaki et al., 1996). The expressed in human pancreatic cancer. In this study the FGF-5 gene, located on chromosome 4q11 (Nguyen et role of FGF-5 in human pancreatic cancer was al., 1988), was originally discovered as a human investigated, as FGF-5 has a classical signal sequence oncogene which had acquired transforming potential for secretion not found in FGF-1 or -2. Northern blot by DNA rearrangement accompanying transfection of analysis with a 306 bp FGF-5 cDNA revealed the NIH3T3 cells with human tumor DNA (Zhan et al., presence of 4.0 kb and 1.6 kb FGF-5 mRNA transcripts 1987). Molecular cloning revealed that the oncogene in both normal and cancerous pancreatic tissues. encoded for a glycosylated 267 amino acid protein Densitometric analysis indicated that 4.0 kb and 1.6 kb (Zhan et al., 1988). Unlike the prototypical FGFs, FGF-5 mRNA transcripts levels were increased 2.4- and FGF-1 and FGF-2, FGF-5 contains a hydrophobic N- 2.7-fold in the cancers by comparison with normal terminal leader sequence typical for eciently secreted tissues, respectively (P50.002, P50.0001). Immunohis- proteins (Zhan et al., 1988), suggesting that this factor tochemistry and in situ hybridization demonstrated that may have an important role in autocrine and paracrine FGF-5 localized in the cancer cells, stromal ®broblast mediated events. and in¯itrating macrophages. FGF-5 mRNA was also The signaling of the FGFs is mediated by a dual- detected in COLO-357 human pancreatic cancer cells. receptor system, consisting of four high-anity Furthermore, secreted FGF-5 protein was present in tyrosine kinase receptors, termed ®broblast growth conditioned medium of COLO-357 cells. Exogeneous factor receptors (FGFRs), and a number of low-anity FGF-5 (0.37 nM) increased the growth of COLO-357 heparan sulfate proteoglycan receptors (Klagsburn and cells by 48% (P50.0001) and increased mitogen- Baird, 1991; Givol and Yayon, 1992; Johnson and activated protein kinase activity. COLO-357 cells Williams, 1993; Fernig and Gallagher, 1994; Mason, expressed the IIIc isoform of the type I FGF receptor, 1994; Burgess and Winkles, 1996). FGFRs are the preferred FGF receptor for FGF-5. These observa- glycoproteins composed of usually three extracellular tions suggest that FGF-5 may participate in autocrine immunoglobulin (Ig)-like domains, a hydrophobic and paracrine pathways promoting pancreatic cancer cell transmembrane region and a cytoplasmic domain that growth in vivo. contains a tyrosine kinase catalytic domain. Alternative mRNA splicing mechanisms result in receptor isoforms Keywords: ®broblast growth factor-5; ®broblast growth that display unique ligand binding properties (Givol factor receptor splice variant; in situ hybridization; and Yayon, 1992; Johnson and Williams, 1993). mitogenic signaling; pancreatic cancer; protein kinases Ligand interaction with FGFRs also requires the presence of endogenous heparan sulfate proteoglycan or exogenous heparin (Givol and Yayon, 1992) and results in oligomerization, activation of the cytoplasmic Introduction receptor tyrosine kinase and receptor autophosphory- lation (Jaye et al., 1992). Subsequent signaling is Fibroblast growth factor-5 (FGF-5) belongs to a group mediated through a number of substrates for tyrosine of homologous heparin-binding polypeptides that are phosphorylation (Ullrich and Schlessinger, 1990), involved in various biological processes including including mitogen-activated protein kinases (MAPKs) development, morphogenesis, tissue growth, and or ERK-1, -2 and -3 (Mason, 1994; Friesel and repair (Fernig and Gallagher, 1994; Burgess and Maciag, 1995). Winkles, 1996). FGFs are angiogenic and mitogenic, Previously, we have demonstrated that FGF-1, and have been implicated in a variety of human FGF-2 and FGF-7 are overexpressed in human neoplasms (Basilico and Moscatelli, 1992; Burgess and pancreatic cancer and that overexpression of FGF-2 Winkles, 1996). The FGF family consists presently of correlates with shorter post-operative survival (Yama- at least ten members, acidic FGF (aFGF or FGF-1), naka et al., 1993; Siddiqi et al., 1995). Moreover, the basic FGF (bFGF or FGF-2), FGF-3 (int-2), FGF-4 human pancreas is rich in heparan sulfate (Gambarini (hst/kFGF), FGF-5, FGF-6, keratinocyte growth et al., 1993). Despite these observations, nothing is factor (KGF or FGF-7), androgen-induced growth known about FGF-5 expression and localization in factor (AIGF or FGF-8), glia-activating factor (GAF human pancreatic tissues. Furthermore, its mitogenic potential in pancreatic cancer cells has not been examined. Therefore, the aim of this study was to Correspondence: M Korc characterize FGF-5 expression in normal and cancer- Received 1 April 1997; revised 27 May 1997; accepted 27 May 1997 ous pancreatic tissues and to investigate its eect on Fibroblast growth factor-5 in human pancreatic cancer MKornmannet al 1418 growth and mitogenic signaling in cultured human FGF-5 immunoreactivity was observed in the cyto- pancreatic cancer cell line. plasm of many cancer cells (Figure 4a) and faint immunoreactivity was present in the ®broblasts (Figure 4a) adjacent to the tumor cells and islet cells (not Results shown). The most intense staining was seen in in®ltrating macrophages (arrowheads). Vascular Expression of FGF-5 mRNA in pancreatic tissues smooth muscle cells were also faintly stained by FGF-5 (not shown). To exclude the possibility of FGF-5 mRNA expression in total RNA from 14 non-speci®c staining by intrinsic peroxidase, we also normal and 16 cancerous human pancreatic tissue used alkaline phosphatase labeled streptavidin and new samples was investigated by Northern blotting using a fuchsin instead of the peroxidase labeled streptavidin 306 bp BamHI/HindIII human FGF-5 cDNA fragment and DAB system. Similar results for FGF-5 staining generated by RT ± PCR from human placenta RNA. were obtained by both methods (not shown). The analysis indicated that FGF-5 mRNA levels were relatively low and hardly detectable in the normal In situ hybridization of FGF-5 in pancreatic tissues pancreas, whereas two transcripts (approximately 4.0 and 1.6 kb) were detectable in many cancer samples In view of the heterogenous populations of cells in the (Figure 1). The 4.0 kb FGF-5 mRNA moiety was normal and cancerous tissues we next carried out in detectable on the original autoradiograph in 12 of 16 situ hybridization analysis. In the normal pancreas, a cancers (75%) and in seven of 14 normal tissues (50%). faint FGF-5 mRNA in situ hybridization signal was The 1.6 kb FGF-5 mRNA moiety was detectable in 10 present in ductal and islet cells (data not shown). In the of 16 cancers (63%) and in four of 14 normal tissues cancer tissues (Figure 4b), a moderate to strong FGF-5 (29%). A larger band (approximately 6 kb) of mRNA in situ hybridization signal was evident in many unknown signi®cance was visible in some of the cancer cells, macrophages (arrowheads) and some lanes. FGF-5 mRNA transcripts were below the level ®broblasts adjacent to the cancer cells. In situ of detection on the original autoradiographs in four hybridization with a sense FGF-5 probe did not cancers and eight normal tissues. The median values produce any speci®c signal (Figure 4c). for FGF-5 expression in the normal tissues of the 4.0 kb and 1.6 kb moieties, determined by densitome- Expression of FGF-5 and FGF receptors in COLO-357 try, were 0.117 and 0.073, respectively, and in the cells cancer tissues 0.280 and 0.195, respectively. Overall, there were 2.4-fold and 2.7-fold increases in the FGF-5 Northern blotting of total RNA prepared from COLO- levels by comparison with the corresponding levels in 357 cells revealed a 4.0 kb FGF-5 mRNA (Figure 5). A normal tissues (P50.002; P50.0001). The relative second 1.6 kb FGF-5 transcript was only detectable on values for all the samples with their corresponding median values are plotted in Figure 2. Immunohistochemistry of FGF-5 in pancreatic tissues To localize FGF-5 protein in pancreatic tissues, immunostaining with highly speci®c anti-human FGF-5 antibodies was carried out. In the normal pancreas (Figure 3a), moderate FGF-5 immunoreac- tivity was present in the ductal cells and faint immunoreactivity was present in the islet cells (out- lined by arrowheads). In the cancer tissues, moderate normal cancer — 28S — 18S — 7S Figure 2 Relative FGF-5 mRNA expression in normal (open Figure 1 Expression of FGF-5 mRNA in six normal and nine triangles, n=14) and cancerous (closed triangles, n=16) human cancerous human pancreatic tissues. Northern blotting of total pancreatic tissue samples. Autoradiographs of Northern blots for RNA (20 mg/lane) was carried out using a human 306 bp BamHI/ FGF-5 and 7S were analysed by densitometry and the level of HindIII FGF-5 cDNA fragment, randon-primed labeled with FGF-5 expression was calculated as the ratio of FGF-5 and 7S, 32 a- P-dCTP (750 000 c.p.m./ml; 14 day exposure). A mouse 7S which served as loading control. The median FGF-5 scores cDNA probe, cross-reactive with human 7S, was used as a (indicated as dotted lines) of normal and cancerous tissue diered loading control (40 000 c.p.m./ml; 6 h exposure).